Automated Affinity Capture and On-Tip Digestion to Accurately Quantitate in Vivo Deamidation of Therapeutic Antibodies

Abstract

Deamidation of therapeutic antibodies may result in decreased drug activity and undesirable changes in pharmacokinetics and immunogenicity. Therefore, it is necessary to monitor the deamidation levels [during storage] and after in vivo administration. Because of the complexity of in vivo samples, immuno-affinity capture is widely used for specific enrichment of the target antibody prior to LC-MS. However, the conventional use of bead-based methods requires large sample volumes and extensive processing steps. Furthermore, with automation difficulties and extended sample preparation time, bead-based approaches may increase artificial deamidation. To overcome these challenges, we developed an automated platform to perform tip-based affinity capture of antibodies from complex matrixes with rapid digestion and peptide elution into 96-well microtiter plates followed by LC-MS analysis. Detailed analyses showed that the new method presents high repeatability and reproducibility with both intra and inter assay CVs < 8%. Using the automated platform, we successfully quantified the levels of deamidation of a humanized monoclonal antibody in cynomolgus monkeys over a time period of 12 weeks after administration. Moreover, we found that deamidation kinetics between in vivo samples and samples stressed in vitro at neutral pH were consistent, suggesting that the in vitro stress test may be used as a method to predict the liability to deamidation of therapeutic antibodies in vivo.

Macro- and Micro-Heterogeneity of Natural and Recombinant IgG Antibodies

“…Asparagine (Asn) deamidation is almost a ubiquitous modification of mAbs and has been well studied because of its contribution to heterogeneity, and its potential impact on potency and immunogenicity. Asn residues in the complementarity determining regions (CDRs) are inherently susceptible to deamidation because of their relatively higher flexibility and exposure to solvents than at other locations [2,18,19,20,21,22]. Deamidation in CDRs can cause a substantial loss of potency [20,22,23,24].…”

“…Deamidation does not impact in vivo clearance [21,26]. Deamidation of Asn residues continues to occur in vivo in the CDRs [19,21,33,34] and Fc region [26] of mAbs. The in vivo deamidation kinetics can be fully predicted via in vitro stress studies under physiological conditions [19,26], which indicates the same non-enzymatic mechanism.…”

Mechanisms of Deamidation of Asparagine Residues and Effects of Main-Chain Conformation on Activation Energy

“…Furthermore, Asn deamidation plays a key role in quality control of antibody drugs. Deamidations have been reported to occur in complementarity-determining regions (CDRs) and Fc regions of recombinant monoclonal antibodies and human endogenous IgG in vivo [ 23 , 24 , 25 , 26 , 27 ]. In particular, antigen-binding affinities are reduced by Asn deamidation in CDRs.…”

High-throughput screening of antibody variants for chemical stability: identification of deamidation-resistant mutants

“…21 However, care must be taken to avoid introduction of artifacts during sample processing, 22-24 and, although high-throughput (HTP) approaches have been reported, 24 these methods are not currently routine and require substantial automation, as well as MS instrumentation and expertise. Alternative HTP methods for deamidation detection are typically indirect, for example, reverse-phase HPLC to detect changes in polarity and hydrophobicity, and ion exchange chromatography or electrophoresis based methods to detect changes in charge.…”